Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.
Mutant= 121 bp
Wild Type = 120 bp
>chr11:69817135+69817254 120bp CCAGCTTCCTCGCTCCAT GTTCTCTCTAATTCACAAGCAGAGC
Wt Sequence:tttgggatttttcagaccgagcagaTTGcttagtgaggtgaggtggcccggaattgggaagcccagcttcctcgctccatgggagcgttccacacgtggactgttattgttgcatgtaagggtgattttttacccagaaaagccaCACATTtgggtggctctgcttgtgaattagagagaactcaaaggcagcaggttccgtgaggaccagaggcttttatgggacagatgtgtaggacagagatggttaggcggccagccaggctcttaaattagatatttggagcagttgtgggaactgtcccaaag
Mutant Sequence: GCAGATTGCTTAGTGAGGTGAGGTGGCCCGGAATTGGGAAGCCCAGCTTCCTCGCTCCATGGGAGCGTTCCACACGTGGACTGTTATTGTTGCATGTAAGGGTGATTTTTTACCCAGAAAAGCCAatcgaattccgaagttcctattctctagaaagtataggaacttcatcagtcaggtacataatatAACTTCGTATAATGTATGCTATACGAAGTTATtaggtggatccactagttctagagcggccTGGGTGGCTCTGCTTGTGAATTAGAGAGAACaCAAAGGCAGCAGGTTCCGTGAGGACCAGAGGCTTTTATGGGACAGATGTGTAGGACAGAGATGGTTAGGCGGCCAGCCAGGCTCTTAAATTAGATATTTGGAGCAGTTGTGGGAACTGTCCCAAAGGGACTTTGGAGTGGTACTTTGAGG
Primer | 5' Label | Sequence 5' → 3' | 3' Label | Primer Type | Reaction | Note |
---|---|---|---|---|---|---|
22365 | AGG AAC TTC ATC AGT CAG GTA CA | Mutant Forward | A | |||
38708 | GTT CTC TCT AAT TCA CAA GCA GAG C | Common | A | |||
38709 | CCA GCT TCC TCG CTC CAT | Wild type Forward | A | |||
38710 | Fluorophore-1 | AGG TGG ATC CAC TAG TTC TAG AGC GG | Quencher-1 | MUT Probe | ||
38711 | Fluorophore-2 | CTG TTA TTG TTG CAT GTA AGG GTG A | Quencher-2 | WT Probe |
Component | Final Concentration |
---|---|
Kapa Probe Fast QPCR | 1.00 X |
ddH2O | |
22365 | 0.40 uM |
38708 | 0.40 uM |
38709 | 0.40 uM |
Wt Probe | 0.15 uM |
Mutant Probe | 0.15 uM |
DNA |
Step | Temp °C | Time | Note |
---|---|---|---|
1 | 95.0 | -- | |
2 | 95.0 | -- | |
3 | 60.0 | -- | |
4 | -- | repeat steps 2-3 for 40 cycles | |
5 | 40.0 | -- | Forever |